Free fatty acids induce endothelial dysfunction and activate protein kinase C and nuclear factor-κB pathway in rat aorta

Background

Insulin resistance is associated with an inappropriate elevation of plasma free fatty acids (FFAs) and endothelial dysfunction. In this study, we asked if elevated circulating FFA levels led to impaired insulin signaling and endothelial dysfunction in-vivo via activation of PKC-mediated inflammatory pathways.

Methods

Sprague-Dawley (S-D) rats were infused with 1) 20% intralipid + heparin (FFA group) or 2) saline alone (Control group) for 6 h. The intact aorta thoracica and aorta abdominalis were then removed. Aortic rings were isolated and evaluated for endothelial-dependent and non-dependent relaxation in an organ bath. The activities of eNOS and PKC were measured in endothelial homogenates prepared from endothelial cells harvested from the aorta. The expression levels of insulin signaling molecules IRS-1, Akt, eNOS, ERK1/ERK2, PKC-α, NFκB-p65 subunit and IκB-α in rat aortic endothelium were determined by immunohistochemistry and Western blot.

Results

Elevation of FFAs resulted in a 35.9% reduction in the response to acetylcholine (p < 0.01), a 26% decline in plasma NOx levels (p < 0.05), a 53% decrease in eNOS activity and a 34 ± 9% inhibition in IRS-1 tyrosine phosphorylation (p < 0.05). We also found a 46% decrease in Akt phosphorylation and a 36% decrease in eNOS phosphorylation. FFA-induced endothelial insulin resistance was associated with 82% increase in total membrane-associated PKC activity, a 1.7-fold increase in total PKC-α protein, 1.29-fold decrease in IκB-α expression levels and 1.47-fold increase in NF-κB p65 subunit expression in rat aortic endothelium.

Conclusion

The molecular mechanisms underlying FFA-induced endothelial insulin resistance and eNOS inhibition may provide an important link implicating the PKC and IκB-α/NF-κB pathways in FFA-mediated inhibition of vascular insulin signaling.     

Role of nuclear factor-κB,reactive oxygen species and cellular signaling in the early phase of acute pancreatitis

Objective Increased knowledge of regulation of signaling proteins in acute pancreatitis (AP) could potentially contribute to the development of novel agents targeted at regulation of cellular signaling. Material and methods Severe AP was induced by administration of 5% sodium taurodeoxycholate in rats. Thirty minutes prior to induction of AP, the animals had an intraperitoneal injection of the antioxidant, N-acetylcysteine (NAC; 10 mg/kg), the NF-κB inhibitor, pyrrolidine dithiocarbamate (PDTC; 100 mg/kg), the ERK inhibitor, PD-98059 (1 mg/kg), or the tyrosine kinase inhibitor, Genistein (1 mg/kg). Plasma levels of IL-6 and IL-10 were determined by ELISA and myeloperoxidase (MPO) levels were measured in the pancreas and lungs 3 and 6 h after sham operation or induction of AP. Results AP results in significant increases in plasma levels of IL-6 at 6 h and IL-10 at 3 and 6 h. Plasma levels of IL-6 were significantly decreased after administration of NAC. NAC pretreatment also increased the ratio of IL-10/IL-6. MPO levels in the pancreas (at 3 and 6 h) and lungs (3 h) were significantly increased in animals with pancreatitis. Pretreatment with NAC, PDTC, PD98059 or Genistein significantly decreased MPO levels in the pancreas at 3 and 6 h and following the administration of PD-98059 or NAC at 6 h. Pretreatment with NAC significantly decreased MPO levels in the lungs at 3 h. Conclusions Pretreatment with NAC could regulate the pro-and anti-inflammatory cytokine balance, probably through NF-κB and ROS signaling pathways. The regulation of signaling pathways during the sequestration of neutrophils in acute pancreatitis seems to vary between organs.     

The inhibition in tumor necrosis factor-α-induced attenuation in endothelial thrombomodulin expression by carvedilol is mediated by nuclear factor-κB and reactive oxygen species

Carvedilol, a nonselective β-adrenoceptor antagonist, has been shown to possess antioxidant effects and reduce the risk of hospitalization and death in patients with severe congestive heart failure, which is featured by the activation of pro-inflammatory cytokines, including tumor necrosis factor-α (TNF-α), and leads to thrombotic complications. Thrombomodulin (TM) plays protective roles against thrombosis. Treatment of ECs with TNF-α resulted in a down-regulation in the TM expression in a time-dependent manner. Pre-treatment of ECs with carvedilol (1 and 10 μM) for 1 h significantly up-regulated the TM expression in ECs in response to TNF-α. When ECs were pre-treated with a nuclear factor-κB (NF-κB) inhibitor, i.e., parthenolide, their TNF-α-mediated down-regulation of TM expression was inhibited. Pre-treatment of ECs with carvedilol inhibited the NF-κB-DNA binding activity in ECs induced by TNF-α. Our findings provide insights into the mechanisms by which carvedilol exerts anti-thrombotic effects by inducing TM expression in ECs in response to pro-inflammatory stimulation.     

BAY 11-7082, a nuclear factor-κB inhibitor,induces apoptosis and S phase arrest in gastric cancer cells

Background

Inhibitors of nuclear factor (NF)-κB pathway have shown potential anti-tumor activities. However, it is not fully elucidated in gastric cancer.

Methods

Firstly, we screened the inhibitory effect of pharmacologic NF-κB inhibitors on cell viability of human gastric cancer cells via CCK-8 assay. Next, cell apoptosis, cell cycle distribution, and mitochondrial membrane potential after BAY 11-7082 treatment were detected by annexin V staining, propidium iodide staining, TUNEL, and JC-1 assays in human gastric cancer HGC-27 cells. Expression of regulatory factors for apoptosis and cell cycle were measured by western blot. Finally, human gastric cancer xenograft model was established to verify the anti-tumor effects of BAY 11-7082 in vivo. Cellular apoptosis and growth inhibition in subcutaneous tumor section were detected by TUNEL and immunohistochemistry assays.

Results

BAY 11-7082 exhibited rapid and potent anti-tumor effects on gastric cancer cells in vitro within a panel of NF-κB inhibitors. BAY 11-7082 induced rapid apoptosis in HGC-27 cells through activating the mitochondrial pathway, as well as down-regulation of Bcl-2 and up-regulation of Bax. BAY 11-7082 also induced S phase arrest through suppressing Cyclin A and CDK-2 expression. Xenograft model confirmed the anti-tumor effects of BAY 11-7082 on apoptosis induction and growth inhibition in vivo.

Conclusions

Our results demonstrated that BAY 11-7082 presented the most rapid and potent anti-tumor effects within a panel of NF-κB inhibitors, and could induce cellular apoptosis and block cell cycle progression both in vitro and in vivo, thus providing basis for clinical application of BAY 11-7082 in gastric cancer cases.     

Cocaine, not morphine, causes the generation of reactive oxygen species and activation of NF-κB in transiently cotransfected heart cells

This study was designed to determine levels of NF-κB reporter gene activity and free radical generation in cultured striated myocytes (H9C2 cells) exposed to cocaine or morphine in the presence of free radical scavengers. Cells were transiently transfected with a NF-κB reporter gene and changes in luciferase activity were detected, by bioluminescence. Using confocal microscopy and 2′,7′-dichlorofluorescin diacetate, cocaine-induced or morphine-induced free radicals were quantified in H9C2 cells. Cocaine and morphine (0–1×10⁻² M) were tested separately. Cocaine but not morphine significantly activated Nf-κB reporter gene, activity in H9C2 cells. Overexpression of IκB inhibited NF-κB reporter activity at low (1×10⁻⁴ M) but not high (1×10⁻² M) cocaine concentrations. Free radicals were generated in H9C2 cells stimulated with cocaine but not with morphine. The production of free radicals and NF-κB reporter gene activity could be blocked with N-acetylcysteine, glutathione, and to a lesser extent, lipoic acid. The results suggest that cocaine induces free radical production, which leads to the activation of NF-κB signal transduction and possible inflammatory responses.     

Involvement of soluble receptor activator of nuclear factor-κB ligand (sRANKL) in collagenase-induced murine osteoarthritis and human osteoarthritis

Joint destruction and excessive bone formation are associated with high expression of soluble receptor activator of nuclear factor-κB ligand (sRANKL). This study was undertaken to investigate the role of sRANKL in collagenase-induced osteoarthritis (CIOA) in mice and in patients with osteoarthritis (OA). The initial phase of CIOA was associated with severe proteoglycan depletion, decreased collagen density, and up-regulation of bone morphogenetic protein (BMP)-2. At the late stage of CIOA, bone remodeling was related with increased BMP2 and RANKL expression in the joints, high sRANKL, and decreased number of activated neutrophils in synovium. CIOA mice showed elevated plasma level of sRANKL but low RANKL expression on blood neutrophils. The percentage of RANKL-positive blood neutrophils was higher in patients with OA than in healthy individuals. Our data indicate that increased local and systemic levels of soluble RANKL might be indicative for OA disorders in mouse and human.     

Diallyl trisufide (DATS) suppresses high glucose-induced cardiomyocyte apoptosis by inhibiting JNK/NFκB signaling via attenuating ROS generation

Background

Hyperglycemia is an important risk factor for cardiovascular diseases no matter if it resulted from type I or type II diabetes mellitus. High glucose-induced generation of reactive oxygen species (ROS) can lead to diabetic cardiomyopathy. In our previous study, we showed that NADPH oxidase-related ROS-induced apoptosis is mediated via the JNK-dependent activation of NF-κB in cardiomyocytes exposed to high glucose (HG).

Objective

In this study, we investigated the mechanisms governing the anti-apoptotic effect of diallyl trisulfide (DATS) on HG-exposed cardiac cells both in vitro and in vivo.

Methods

H9c2 cells were incubated with media containing 5.5 or 33 mM of glucose for 36 h in the presence or absence of DATS.

Results

We found that DATS treatment led to a dose-dependent decrease in ROS levels as well as protein levels of p22phox, gp91phox, phosphorylated JNK, and phosphorylated c-Jun. In addition, DATS inhibited the HG-induced activation of caspase 3 as well as the nuclear translocation of NF-κB. Similar results were observed in HG-exposed neonatal primary cardiomyocytes and streptozotocin-treated diabetic rats. Echocardiographic data showed that DATS administration led to a marked increase in fractional shortening and cardiac output.

Conclusion

DATS appears to suppress high glucose-induced cardiomyocyte apoptosis by inhibiting NADPH oxidase-related ROS and its downstream JNK/NF-κB signaling, and may possess the potential on the therapy of diabetic cardiomyopathy.     

Autophagy plays a protective role in advanced glycation end products-induced apoptosis of chondrocytes via regulation of tumor necrosis factor-α,nuclear factor-κ B and reactive oxygen species

Objective: To study the adverse effects of advanced glycation end products(AGEs) on chondrocytes and the role of autophagy in this process. Methods: Chondrocytes were harvested from the human articular cartilage tissues in surgery. AGEs were administered during chondrocytes culture. The rapamycin was used to induce autophagy. The cell viability was determined by 3-[4,5-dimethylthiazol2-yl]-2,5-diphenyl tetrazolium bromide(MTT) assay.The expression of tumor necrosis factor-α(TNF-α) and nuclear factor-κ B(NF-κ B) was detected by quantitative real-time polymerase chain reaction. The reactive oxygen species(ROS) production and apoptosis of the chondrocytes were determined by fluorescent probe and flow cytometer, respectively. Results: The chondrocytes viability was significantly reduced after 12 h incubation with AGEs(P0.01)). In contrast, rapamycin pretreatment increased the chondrocytes viability through autophagy. AGEs increased TNF-α and NF-κ B mRNA expression of chondrocytes and autophagy receded or proceeded the change. AGEs increased intracellular ROS accumulation and autophagy reversed the change. AGEs accelerated chondrocytes apoptosis and autophagy suspended apoptosis. Conclusions: Accumulation of AGEs may have an adverse role for chondrocytes by increasing TNF-α and NF-κB expression, ROS accumulation and apoptosis; meanwhile, autophagy ameliorates the AGEsinduced adverse effects.     

Role of rivaroxaban in sunitinib-induced renal injuries via inhibition of oxidative stress-induced apoptosis and inflammation through the tissue nacrosis factor-α induced nuclear factor-κappa B signaling pathway in rats

Journal of Thrombosis and Thrombolysis - Rivaroxaban (RIVA) inhibits factor Xa and exhibits antithrombotic and anti-inflammatory activities by inhibiting several cellular signaling molecules....     

Role of atypical protein kinase C in activation of sterol regulatory element binding protein-1c and nuclear factor kappa B (NFκB) in liver of rodents used as a model of diabetes, and relationships to hyperlipidaemia and insulin resistance

Aims/hypothesis  Previous findings in rodents used as a model of diabetes suggest that insulin activation of atypical protein kinase C (aPKC) is impaired in muscle, but, unexpectedly, conserved in liver, despite impaired hepatic protein kinase B (PKB/Akt) activation. Moreover, aPKC at least partly regulates two major transactivators: (1) hepatic sterol receptor binding protein-1c (SREBP-1c), which controls lipid synthesis; and (2) nuclear factor kappa B (NFκB), which promotes inflammation and systemic insulin resistance. Methods  In Goto–Kakizaki rats used as a model of type 2 diabetes, we examined: (1) whether differences in hepatic aPKC and PKB activation reflect differences in activation of IRS-1- and IRS-2-dependent phosphatidylinositol 3-kinase (PI3K); (2) whether hepatic SREBP-1c and NFκB are excessively activated by aPKC; and (3) metabolic consequences of excessive activation of hepatic aPKC, SREBP-1c and NFκB. Results  In liver, as well as in muscle, IRS-2/PI3K activation by insulin was intact, whereas IRS-1/PI3K activation by insulin was impaired. Moreover, hepatic IRS-2 is known to control hepatic aPKC during insulin activation. Against this background, selective inhibition of hepatic aPKC by adenoviral-mediated expression of mRNA encoding kinase-inactive aPKC or short hairpin RNA targeting Irs2 mRNA and partially depleting hepatic IRS-2 diminished hepatic SREBP-1c production and NFκB activities, concomitantly improving serum lipids and insulin signalling in muscle and liver. Similar improvements in SREBP-1c, NFκB and insulin signalling were seen in ob/ob mice following inhibition of hepatic aPKC. Conclusions/interpretation  In diabetic rodent liver, diminished PKB activation may largely reflect impaired IRS-1/PI3K activation, while conserved aPKC activation reflects retained IRS-2/PI3K activity. Hepatic aPKC may also contribute importantly to excessive SREPB-1c and NFκB activities. Excessive hepatic aPKC-dependent activation of SREBP-1c and NFκB may contribute importantly to hyperlipidaemia and systemic insulin resistance. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorised users.     

Effects of smoking cessation on expressions of nuclear factor-κB, matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 in airway epithelial cells of smoking rats

Objective To investigate the effects of smoking and smoking cessation on airway inflammation and remodeling in chronic obstructive pulmonary diseases through detecting mRNA and protein expressions of nuclear factor-κB (NF-κB), cell matrix metalloproteinase-9 (MMP-9) and cellular tissue inhibitor of metalloproteinase-1 (TIMP-1) in airway epithelial cells of smoking and smoking cessation rats. Methods Twenty-four male Wistar rats were randomly divided into control group, smoking group and smoking cessation group,eight in each group. Hybridization in situ and immunohistochemistry were used to detect mRNA and protein expressions of NF-κB, MMP-9 and TIMP-1 in airway epithelial cells of rats. Results ① Compared with control group (0.29 ± 0.06,0.29±0.06), mRNA and protein expressions of NF-κB in smoking group (0.45±0.04,0.41±0.03) and smoking cessation group (0.40±0.05,0.37±0.03) were higher (all P＜0.05). The mRNA and protein expressions of NF-κB in smoking cessation group were lower than those in smoking group (all P ＜0.05). ②Compared with control group (0.30±0.06,0.30±0.06) ,mRNA and protein expressions of MMP-9 in smoking group (0.52±0.03,0.51±0.07) and smoking cessation group (0.38±0.03,0.33±0.02) were higher (all P＜0.05). The mRNA and protein expressions of MMP-9 in smoking cessation group were lower than those in smoking group (all P＜0.05). ③Compared with control group (0.26±0.04, 0.26±0.04), mRNA and protein expressions of TIMP-1 in smoking group (0.49±0.05,0.37±0.03) and smoking cessation group (0.42±0.04,0.35±0.03) were higher (all P ＜0.05). The mRNA and protein expressions of TIMP-1 in smoking cessation group were lower than those in smoking group (all P ＜ 0.05). ④ Compared with control group (1.00±0.02,1.00±0.02), MMP-9/TIMP-1 mRNA and protein expressions were larger than one in smoking group (1.07±0.14, 1.37±0.19), and less than one in smoking cessation group (0.92±0.13,0.94±0.10) (all P ＜0.05). ⑤The mRNA and protein expressions of NF-κB and MMP-9in each group were positively correlation (r=0.87,0.66,all P ＜0.05). Conclusions In airway epithelial cells of smoking rats, mRNA and protein expressions of NF-κB, MMP-9 and TIMP-1 increase, and MMP-9/TIMP-1 is larger than one. After stoping smoking, mRNA and protein expressions of NF-κB,MMP-9 and TIMP-1 decrease, and MMP-9/TIMP-1 is less than one. This experiment explains that smoking can cause airway inflammation and remodeling, smoking cessation can reduce airway inflammation and remodeling.     

Effects of smoking cessation on expressions of nuclear factor-κB, matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 in airway epithelial cells of smoking rats

Objective To investigate the effects of smoking and smoking cessation on airway inflammation and remodeling in chronic obstructive pulmonary diseases through detecting mRNA and protein expressions of nuclear factor-κB (NF-κB), cell matrix metalloproteinase-9 (MMP-9) and cellular tissue inhibitor of metalloproteinase-1 (TIMP-1) in airway epithelial cells of smoking and smoking cessation rats. Methods Twenty-four male Wistar rats were randomly divided into control group, smoking group and smoking cessation group,eight in each group. Hybridization in situ and immunohistochemistry were used to detect mRNA and protein expressions of NF-κB, MMP-9 and TIMP-1 in airway epithelial cells of rats. Results ① Compared with control group (0.29 ± 0.06,0.29±0.06), mRNA and protein expressions of NF-κB in smoking group (0.45±0.04,0.41±0.03) and smoking cessation group (0.40±0.05,0.37±0.03) were higher (all P＜0.05). The mRNA and protein expressions of NF-κB in smoking cessation group were lower than those in smoking group (all P ＜0.05). ②Compared with control group (0.30±0.06,0.30±0.06) ,mRNA and protein expressions of MMP-9 in smoking group (0.52±0.03,0.51±0.07) and smoking cessation group (0.38±0.03,0.33±0.02) were higher (all P＜0.05). The mRNA and protein expressions of MMP-9 in smoking cessation group were lower than those in smoking group (all P＜0.05). ③Compared with control group (0.26±0.04, 0.26±0.04), mRNA and protein expressions of TIMP-1 in smoking group (0.49±0.05,0.37±0.03) and smoking cessation group (0.42±0.04,0.35±0.03) were higher (all P ＜0.05). The mRNA and protein expressions of TIMP-1 in smoking cessation group were lower than those in smoking group (all P ＜ 0.05). ④ Compared with control group (1.00±0.02,1.00±0.02), MMP-9/TIMP-1 mRNA and protein expressions were larger than one in smoking group (1.07±0.14, 1.37±0.19), and less than one in smoking cessation group (0.92±0.13,0.94±0.10) (all P ＜0.05). ⑤The mRNA and protein expressions of NF-κB and MMP-9in each group were positively correlation (r=0.87,0.66,all P ＜0.05). Conclusions In airway epithelial cells of smoking rats, mRNA and protein expressions of NF-κB, MMP-9 and TIMP-1 increase, and MMP-9/TIMP-1 is larger than one. After stoping smoking, mRNA and protein expressions of NF-κB,MMP-9 and TIMP-1 decrease, and MMP-9/TIMP-1 is less than one. This experiment explains that smoking can cause airway inflammation and remodeling, smoking cessation can reduce airway inflammation and remodeling.     

Soluble receptor activator of nuclear factor-κB ligand (RANKL) and osteoprotegerin in ankylosing spondylitis: OPG is associated with poor physical mobility and reflects systemic inflammation

The objective of the study was to investigate the role of receptor activator of nuclear factor-κB ligand (RANKL) and osteoprotegerin (OPG) in ankylosing spondylitis (AS). Serum levels of soluble RANKL (sRANKL) and OPG were measured in 42 AS patients and 26 healthy controls. We evaluated the AS patient's disease activity, functional ability, global assessment, and physical mobility and tested markers of systemic inflammation, erythrocyte sedimentation rate (ESR), and C-reactive protein (CRP) levels. Serum levels of sRANKL [mean (SD), 4.75 (1.88) vs. 3.70 (1.14) pmol/l, p = 0.015] and OPG [mean (SD), 5.18 (1.19) vs. 4.52 (0.85) pmol/l, p = 0.026] were significantly higher in the 42 AS patients than the 26 healthy controls. Interestingly, serum OPG levels correlated significantly with ESR (r = 0.417, p = 0.007), CRP (r = 0.524, p < 0.001), tragus-to-wall distance (r = 0.556, p < 0.001), fingertip-to-floor distance (r = 0.423, p = 0.007), and occiput-to-wall distance (r = 0.465, p = 0.002) and correlated inversely with modified Schober index (r = −0.525, p = 0.001), cervical rotation (r = −0.403, p = 0.022), lateral lumbar flexion (r = −0.587, p < 0.001), and chest expansion (r = −0.553, p < 0.001). Moreover, in the AS patients with higher (≥4.925 pmol/l, n = 21) serum OPG levels, there were significant increases in the tragus-to-wall distance (p = 0.007), fingertip-to-floor distance (p = 0.023), and CRP levels (p = 0.014) and decreased in the modified Schober index (p = 0.012), lateral lumbar flexion (p = 0.019), and chest expansion (p = 0.005). Serum levels of sRANKL and OPG are increased in the AS patients and may participate in the disease process of AS. Production of OPG has association with poor physical mobility and may reflect systemic inflammation in AS.     

Acetyl-L-carnitine confers neuroprotection against lipopolysaccharide (LPS) -induced neuroinflammation by targeting TLR4/NFκB,autophagy, inflammation and oxidative stress

Acetyl-L-carnitine has been shown to exert neuroprotection against neurodegenerative diseases. The present study was performed to evaluate neuroprotection effects of acetyl-L-carnitine against lipopolysaccharide (LPS) -induced neuroinflammation and clarify possible mechanisms. A single dose (500 µg/kg) of LPS was intraperitoneally injected to rats to induce model. The animals were intraperitoneally treated with different doses of acetyl-L-carnitine (30, 60, and 100) for 6 days. Y-maze task, single-trial passive avoidance and novel object recognition tests were used to evaluate memory impairments. ELISA assay was used to evaluate the expression of TLR4/NFκB, autophagic and oxidative stress markers. Our result showed that intraperitoneal injection of LPS resulted in initiation of neuroinflammation by activation of TLR4/NFκB, suppression of autophagic markers such as LC3 II/ LC3 I ratio and becline-1, and excessive production of ROS and MDA. Intraperitoneal administration of acetyl-L-carnitine contributed to neuroprotection against LPS -induced neuroinflammation by suppression of TLR4/NFκB pathway, restoring activity of autophagy and inhibition of oxidative stress. Collectively, our findings show that acetyl-L-carnitine attenuated LPS-induced neuroinflammation by targeting TLR4/NFκB pathway, autophagy and oxidative stress.

     

Diurnal variation and effect of insulin on circulating high molecular weight (HMW) adiponectin and NF-κB activity in human endothelial cells

ObjectiveTo study the diurnal variation and the effect of insulin on adiponectin multimers and nuclear factor-kappaB (NF-κB) activity in human endothelial cells.Methods and resultsWe utilized a prolonged insulin–glucose infusion in six healthy human subjects. HMW and total adiponectin levels were higher in the morning and lower at night; NF-κB activities in serum treated human microvascular endothelial cells (HMEC-1) cells were lower in the morning and higher at night. Hyperinsulinemic induction significantly decreased HMW and total adiponectin levels but increased NF-κB activity in serum treated HMEC-1 cells (P < 0.05, P < 0.01). There were no significant changes to MMW and LMW adiponectin levels (P > 0.05).ConclusionCircadian rhythm of HMW adiponectin and NF-κB activity are altered by hyperinsulinemia providing novel insights adiponectin and NF-κB biology, which may be pertinent to insulin resistant states, e.g. obesity and type 2 diabetes mellitus.